Dao M. Nguyen

MutT Homolog 1 (MTH1) maintains multiple KRAS-driven pro-malignant pathways

Oncogenic RAS promotes production of reactive oxygen species (ROS), which mediate pro-malignant signaling but can also trigger DNA damage-induced tumor suppression. Thus RAS-driven tumor cells require redox-protective mechanisms to mitigate the damaging aspects of ROS. Here, we show that MutT Homolog 1 (MTH1), the mammalian 8-oxodGTPase that sanitizes oxidative damage in the nucleotide pool, is important for maintaining several KRAS-driven pro-malignant traits in a nonsmall cell lung carcinoma (NSCLC) model. MTH1 suppression in KRAS-mutant NSCLC cells impairs proliferation and xenograft tumor formation. Furthermore, MTH1 levels modulate KRAS-induced transformation of immortalized lung epithelial cells. MTH1 expression is upregulated by oncogenic KRAS and correlates positively with high KRAS levels in NSCLC human tumors. At a molecular level, in p53-competent KRAS-mutant cells, MTH1 loss provokes DNA damage and induction of oncogene-induced senescence. In p53-nonfunctional KRAS-mutant cells, MTH1 suppression does not produce DNA damage but reduces proliferation and leads to an adaptive decrease in KRAS expression levels. Thus, MTH1 not only enables evasion of oxidative DNA damage and its consequences, but can also function as a molecular rheostat for maintaining oncogene expression at optimal levels. Accordingly, our results indicate MTH1 is a novel and critical component of oncogenic KRAS-associated malignancy and its inhibition is likely to yield significant tumor-suppressive outcomes in KRAS-driven tumors.

TRAIL induces autophagic protein cleavage through caspase activation in melanoma cell lines under arginine deprivation

Arginine deprivation is a promising strategy for treating ASS-negative malignant tumors including melanoma. However, autophagy can potentially counteract the effectiveness of this treatment by acting as a pro-survival pathway. By combining tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with arginine deprivation using ADI-PEG20 (pegylated arginine deiminase), we achieved enhanced apoptosis and accelerated cell death in melanoma cell lines. This implies a switch from autophagy to apoptosis. In our current investigation, we found that TRAIL could induce the cleavage of two key autophagic proteins, Beclin-1 and Atg5, in the combination treatment. Using specific inhibitors for individual caspases, we found that caspase-8 inhibitor could completely abolish the cleavage. Furthermore, caspase-8 inhibitor was able to fully reverse the enhanced cytotoxicity induced by TRAIL. Inhibitors for caspase-3, 6, 9, and 10 were able to block the cleavage of these two autophagic proteins to some extent and correspondingly rescue cells from the cytotoxicity of the combination of TRAIL and arginine deprivation. In contrast, calpain inhibitor could not prevent the cleavage of either Beclin-1 or Atg5, and was unable to prevent cell death. Overall, our data indicate that the cleavage of Beclin-1 and Atg5 by TRAIL-initiated caspase activation is one of the mechanisms that lead to the enhancement of the cytotoxicity in the combination treatment.

The combination of ADI-PEG20 and TRAIL effectively increases cell death in melanoma cell lines

Current treatment for advanced, metastatic melanoma is not very effective, and new modalities are needed. ADI-PEG20 is a drug that specifically targets ASS-negative malignant melanomas while sparing the ASS-expressing normal cells. Although laboratory research and clinical trials showed promising results, there are some ASS-negative cell lines and patients that can develop resistance to this drug. In this report, we combined ADI-PEG20 with another antitumor drug TRAIL to increase the killing of malignant melanoma cells. This combination can greatly inhibit cell growth (to over 80%) and also enhanced cell death (to over 60%) in four melanoma cell lines tested compared with control. We found that ADI-PEG20 could increase the cell surface receptors DR4/5 for TRAIL and that caspase activity correlated with the increased cell death. These two drugs could also increase the level of Noxa while decrease that of survivin. We propose that these two drugs can complement each other by activating the intrinsic and extrinsic apoptosis pathways, thus enhance the killing of melanoma cells.

The Hedgehog processing pathway is required for NSCLC growth and survival

Considerable interest has been generated from the results of recent clinical trials using smoothened (SMO) antagonists to inhibit the growth of hedgehog (HH) signaling-dependent tumors. This interest is tempered by the discovery of SMO mutations mediating resistance, underscoring the rationale for developing therapeutic strategies that interrupt HH signaling at levels distinct from those inhibiting SMO function. Here, we demonstrate that HH-dependent non-small cell lung carcinoma (NSCLC) growth is sensitive to blockade of the HH pathway upstream of SMO, at the level of HH ligand processing. Individually, the use of different lentivirally delivered shRNA constructs targeting two functionally distinct HH-processing proteins, skinny hedgehog (SKN) or dispatched-1 (DISP-1), in NSCLC cell lines produced similar decreases in cell proliferation and increased cell death. Further, providing either an exogenous source of processed HH or a SMO agonist reverses these effects. The attenuation of HH processing, by knocking down either of these gene products, also abrogated tumor growth in mouse xenografts. Finally, we extended these findings to primary clinical specimens, showing that SKN is frequently overexpressed in NSCLC and that higher DISP-1 expression is associated with an unfavorable clinical outcome. Our results show a critical role for HH processing in HH-dependent tumors, identifies two potential druggable targets in the HH pathway, and suggest that similar therapeutic strategies could be explored to treat patients harboring HH ligand-dependent cancers.

Differential abundance of CK1a provides selectivity for pharmacological CK1a activators to target WNT-dependent tumors

A small-molecule CK1α activator inhibits the growth of WNT-driven intestinal tumors without toxicity to normal epithelium. Treating WNT-driven tumors The WNT signaling pathway powers the growth of various tumors, particularly colorectal cancer (CRC). However, WNT-targeted inhibitors are very toxic to normal gastrointestinal tissue, precluding their approval for clinical use. Li et al. show that WNT could be targeted by activating a kinase that inhibits the pathway. A small-molecule activator of the kinase CK1α called SSTC3 suppressed WNT activity in CRC cell lines, prevented tumor growth, and increased survival in mouse models of primary and metastatic CRC. Because the effects of SSTC3 were selective to cells with high WNT activity and low CK1α abundance, SSTC3 was minimally toxic to normal gastrointestinal epithelium. Thus, SSTC3 and its future derivatives may be a promising therapeutic for CRC patients. Constitutive WNT activity drives the growth of various human tumors, including nearly all colorectal cancers (CRCs). Despite this prominence in cancer, no WNT inhibitor is currently approved for use in the clinic largely due to the small number of druggable signaling components in the WNT pathway and the substantial toxicity to normal gastrointestinal tissue. We have shown that pyrvinium, which activates casein kinase 1α (CK1α), is a potent inhibitor of WNT signaling. However, its poor bioavailability limited the ability to test this first-in-class WNT inhibitor in vivo. We characterized a novel small-molecule CK1α activator called SSTC3, which has better pharmacokinetic properties than pyrvinium, and found that it inhibited the growth of CRC xenografts in mice. SSTC3 also attenuated the growth of a patient-derived metastatic CRC xenograft, for which few therapies exist. SSTC3 exhibited minimal gastrointestinal toxicity compared to other classes of WNT inhibitors. Consistent with this observation, we showed that the abundance of the SSTC3 target, CK1α, was decreased in WNT-driven tumors relative to normal gastrointestinal tissue, and knocking down CK1α increased cellular sensitivity to SSTC3. Thus, we propose that distinct CK1α abundance provides an enhanced therapeutic index for pharmacological CK1α activators to target WNT-driven tumors.

Arsenic Attenuates GLI Signaling, Increasing or Decreasing its Transcriptional Program in a Context Dependent Manner

The metalloid arsenic is a worldwide environmental toxicant, exposure to which is associated with many adverse outcomes. Arsenic is also an effective therapeutic agent in certain disease settings. Arsenic was recently shown to regulate the activity of the Hedgehog (HH) signal transduction pathway, and this regulation of HH signaling was proposed to be responsible for a subset of arsenic’s biologic effects. Surprisingly, these separate reports proposed contradictory activities for arsenic, as either an agonist or antagonist of HH signaling. Here we provide in vitro and in vivo evidence that arsenic acts as a modulator of the activity of the HH effector protein glioma-associated oncogene family zinc finger (GLI), activating or inhibiting GLI activity in a context-dependent manner. This arsenic-induced modulation of HH signaling is observed in cultured cells, patients with colorectal cancer who have received arsenic-based therapy, and a mouse colorectal cancer xenograft model. Our results show that arsenic activates GLI signaling when the intrinsic GLI activity is low but inhibits signaling in the presence of high-level GLI activity. Furthermore, we show that this modulation occurs downstream of primary cilia, evidenced by experiments in suppressor of fused homolog (SUFU) deficient cells. Combining our findings with previous reports, we present an inclusive model in which arsenic plays dual roles in GLI signaling modulation: when GLIs are primarily in their repressor form, arsenic antagonizes their repression capacity, leading to low-level GLI activation, but when GLIs are primarily in their activator form, arsenic attenuates their activity.

Inhibition of WNT signaling attenuates self-renewal of SHH-subgroup medulloblastoma

The SMOOTHENED inhibitor vismodegib is FDA approved for advanced basal cell carcinoma (BCC), and shows promise in clinical trials for SONIC HEDGEHOG (SHH)-subgroup medulloblastoma (MB) patients. Clinical experience with BCC patients shows that continuous exposure to vismodegib is necessary to prevent tumor recurrence, suggesting the existence of a vismodegib-resistant reservoir of tumor-propagating cells. We isolated such tumor-propagating cells from a mouse model of SHH-subgroup MB and grew them as sphere cultures. These cultures were enriched for the MB progenitor marker SOX2 and formed tumors in vivo. Moreover, while their ability to self-renew was resistant to SHH inhibitors, as has been previously suggested, this self-renewal was instead WNT-dependent. We show here that loss of Trp53 activates canonical WNT signaling in these SOX2-enriched cultures. Importantly, a small molecule WNT inhibitor was able to reduce the propagation and growth of SHH-subgroup MB in vivo, in an on-target manner, leading to increased survival. Our results imply that the tumor-propagating cells driving the growth of bulk SHH-dependent MB are themselves WNT dependent. Further, our data suggest combination therapy with WNT and SHH inhibitors as a therapeutic strategy in patients with SHH-subgroup MB, in order to decrease the tumor recurrence commonly observed in patients treated with vismodegib.

Abstract 1206: Cisplatin resistant non small cell lung cancer is sensitive to arginine deprivation therapy

Drug resistance is a major obstacle to cancer chemotherapy. Despite early positive response to platinum-based chemotherapy, the majority of non small cell lung cancer (NSCLC) develops resistance. We have previously shown that all cisplatin resistant (CR) cell lines exhibit high levels of reactive oxygen species (ROS) production which leads to alteration in tumor metabolism. We and others have shown that CR cells have low or no expression of argininosuccinate synthetase (ASS) when compared to their parental cells. Since ASS is a key enzyme to synthesize arginine from citrulline, lack of ASS expression will make arginine an essential amino acid for these CR tumors. Certain NSCLC cell lines/tumor samples also express very low levels of ASS; however, it is unclear how this expression will be affected by cisplatin treatment. In this report, we investigate the underlying mechanism using 6 pairs of NSCLC cell lines and their CR variants: H460/H460CR, F/FCR, S/SCR, A549/A549CR, H1299/H1299CR, and H23/H23CR. Note that H460, F, S are ASS(+) and A549, H1299, H23 are ASS(-). No ASS expression due to hypermethylation was found in H1299, while in A549 and H23, it is most likely due to transcription regulation which governs ASS expression, as we have been previously reported in melanoma. All these CR cells possess at least 7 fold resistances to cisplatin and express 3-5 fold increase in ROS levels when compared to parental cell counterparts. CR cells, derived from parental cells that possess high ASS expression, exhibited significantly lower levels of ASS once they became resistant to cisplatin. These CR cells were also hypersensitive to arginine deprivation using ADI-PEG20 (provided by Polaris Inc) which degrades arginine to citrulline. Importantly, the addition of 50ng/ml of ADI-PEG20 restored cisplatin sensitivity to CR cells. With the combination of cisplatin and ADI-PEG20, the ID50 dosage of cisplatin decreased (5-7 fold) to the original levels as their parental counterparts. When compared with cisplatin alone treatment, the percentage of cells with caspase activation increased from 5% (2ug/ml of cisplatin) to 70% (combination with ADI-PEG20). However, in three pairs of CR cells derived from ASS(-) cells, the ASS remains negative in H1299CR while in A549CR and H23CR the ASS expression showed 1-1.5 fold increase both at mRNA and protein levels. Nevertheless, the addition of ADI-PEG20 still can sensitize these CR cells to cisplatin treatment. To further verify the role of ASS in cisplatin resistance, we have overexpressed ASS into SCR cell line and determine the sensitivity to cisplatin. This transfectant became resistant to ADI-PEG20, but maintains similar levels of cisplatin resistance. These data suggest that silencing ASS in these CR cells is most likely a consequence of other biochemical changes occurring in CR cells. These metabolic alterations can be used to target CR lung cancer. Supported by Department of Veterans Affairs, CDA2 award (1K2BX001289). Citation Format: Medhi Wangpaichitr, Chunjing Wu, Dao M. Nguyen, Min You, Ying Ying Li, Shumei Chen, Lynn G. Feun, Niramol Savaraj. Cisplatin resistant non small cell lung cancer is sensitive to arginine deprivation therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1206. doi:10.1158/1538-7445.AM2015-1206

Abstract 1435: Exploiting obligate arginine auxotrophy in tumor cells lacking arginino-succinate synthetase (ASS) expression to develop targeted molecular therapy for non-small cell lung cancer (NSCLC)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background. Malignant cells frequently exhibit dysregulation of nutrient/energy metabolism that can be exploited for development of novel targeted molecular therapy. Transcriptional repression of ASS, an enzyme essential for arginine production, in certain cancers and not normal cells makes these tumor cells selectively auxotrophic (dependent on external source) of this semi-essential amino acid and extremely susceptible to arginine depletion. One of the clinically applicable strategies to deplete extracellular arginine is systemic administration of the enzyme arginine di-iminase (ADI) conjugated with polyethylene glycol (PEG20) for optimal bioavailability. While well characterized in malignant pleural mesothelioma, melanoma and hepatocellular carcinoma, the status of ASS expression and sensitivity to ADI-PEG20 has not previously been investigated in NSCLC and this is the overarching objective of this study. Materials and Methods. ASS expression in 12 NSCLC cell lines and normal cells is determined by quantitative RT-PCR, western blots. ASS expression in lung cancers is quantified by immunohistochemistry (IHC) of tissue microarray of 72 NSCLC tumors. ADI-PEG20-mediated inhibition of cell viability, autophagy activation, induction of apoptosis and cell cycle arrest are assayed using MTT, fluorescence microscopy, AnnexinV/PI staining with flow cytometry respectively. Methylation of the promotor region of the ASS gene in ASS-negative cells is evaluated by methylation-specific PCR. Results. Six of 12 NSCLC cells have no or extremely low basal expression of ASS mRNA and protein and are, thus, exquisitely sensitive to ADI-PEG20 treatment (IC50 values: 45± 5 to 90±8 ng/ml (mean ± standard deviation, n=6)). ASS+ NSCLC cells and normal cells (primary or immortalized human fibroblasts) are totally resistant to ADI-PEG20. About 80% of NSCLC tumors do not express ASS by IHC using a well-validated anti-ASS antibody (Santa Cruz, clone H231). ADI-PEG20 treatment strongly activates autophagy with accumulation of autophagic vacuoles detected by fluorescence microscopy. Significant induction of apoptosis (15±3% to 55±6%, n=4) is noted in 5 NSCLC cells treated with ADI-PEG20 (100 or 200 ng/ml for 4 or 7 days) as well as cell cycle arrest. Two cell lines have methylated ASS promotor (one complete, one partial) as the cause of gene silencing. Conclusion. Up to 50% of NSCLC cell lines and 80% of NSCLC tumors lack ASS expression. ASS- NSCLC are selectively and significantly susceptible to ADI-PEG20-mediated arginine depletion in vitro. Induction of autophagy, apoptosis and cell cycle arrest collectively contribute ADI-PEG20-mediated cytotoxicity. As ADI-PEG20 is currently in phase II clinical trials for other carcinomas with encouraging results, it can be used to develop novel targeted molecular therapy for NSCLC tumors lacking ASS. Citation Format: Min You, Medhi Wangpaichitr, Jonathan D. Nguyen, Jennifer R. Chapman, Maureen Cioffi-Lavina, Niramol Savaraj, Dao M. Nguyen. Exploiting obligate arginine auxotrophy in tumor cells lacking arginino-succinate synthetase (ASS) expression to develop targeted molecular therapy for non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1435. doi:10.1158/1538-7445.AM2014-1435

Abstract 1951: Role of embryonic signaling pathways in lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Embryonic signaling pathways that play an important role during embryo development, later on support the maintenance of adult stem cells and are also found activated in various cancers. The activated embryonic signaling, mainly Hedgehog (HH), WNT and NOTCH, have been shown to be activated in a number of cancers and demonstrated to promote growth, provide survival advantage and resistance to various drugs. The role of these embryonic signaling pathways in the development of lung cancer is not well defined. Identification of particular pathways active in cancer and elucidation of their mechanism of action in cancer development could provide a basis of devising a successful therapeutic intervention. Objective: Define the role of embryonic signaling pathways in cancer cells. Methodology: The presence and functional interaction of various embryonic signaling pathways in lung cancer was evaluated using human non-small cell lung carcinoma cell lines (e.g. HOP62, A549, H23, H522) as model system. The signaling pathways were inhibited using specific small molecule inhibitors and shRNAs mediated knockdown of the pathway components. The lentiviruses expressing shRNA were made and used to deliver the shRNA in NSCLC cells. The effect on the target gene expression (quantitative real time RT-PCR and western blotting), cell proliferation (ATP quantitation), cell migration (scratch/wound assay) and anchorage independent growth (soft agar assay), was evaluated. Currently, the activation of pathways in cancer cells of primary lung cancer samples is being evaluated by immunohistochemistry (IHC) and RNA in situ hybridization (ISH). Preliminary Results and Conclusions: Attenuation of individual embryonic signaling pathways using specific small molecule inhibitors and shRNAs, indicated crosstalk and hierarchy of these pathways in controlling various processes in tested cancer cells. Our results suggest the predominance of HH signaling in non-small cell lung carcinoma. We are in the process of expanding our observation to more cell lines and human cancer samples. Our work will establish the role of embryonic signaling pathways in lung cancer and would further help in establishing a framework for future therapeutic intervention. Citation Format: Samer Singh, Dao M. Nguyen, Anthony J. Capobianco, David J. Robbins. Role of embryonic signaling pathways in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1951. doi:10.1158/1538-7445.AM2014-1951